CN101705207B - Culture medium of amniotic fluid cells - Google Patents
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Abstract
The invention relates to a culture medium of amniotic fluid cells, which belongs to the field of biochemistry. The culture medium comprises the following components: basic medium, transferrin, sodium selenite, insulin, triiodothyronine, glucagons, basic fibroblast growth factor, hydrocortisone, testosterone, estradiol, progesterone and added antioxidant, wherein the antioxidant is a mixture of vitamin E and L-ascorbic acid; and the culture medium can be used in amniotic fluid cell culture after 4 to 15 volume percent of fetal calf serum is added in the culture medium, and a great amount of amniotic fluid cells which are at a division stage and meet the requirements of clinical diagnosis and scientific research can be obtained just within 7 to 8 days. The culture medium remarkably improves the culture effects of amniotic fluid cells and obviously increases cloning efficiency and cloning growth rate, reduces culture time, increases the success rate of cell culture, and fully plays the synergetic effects of vitamin E and L-ascorbic acid of different antioxidants.
Description
Technical field
A kind of culture medium of amniotic fluid cells of the present invention belongs to biochemical field.
Background technology
Antenatal diagnosis is eugenic important component part, it is to be based upon on the genetic counseling basis by antenatal detection foetus health situation, can take measures early to the infant of suffering from serious inherited disease, congenital malformation, deleterious gene accumulation among the minimizing crowd, to reduce the infant birth, improve the health of the people, so tool is of great significance.Antenatal diagnosis is pregnant mainly at present that early stage chorionic villi is drawn materials, second trimester amniotic fluid and Cord blood, maternal blood isolation of fetal cells and implant before the methods such as diagnosis, but the inspection of second trimester amniocyte is still topmost method.This is that the amniocentesis inspection has operational safety because compare with other method, and pregnant turnover rate is lower than rate of spontaneous abortion and the comparatively reliable advantage of diagnosis chromosome abnormalty.
The main object of amniocentesis inspection is advanced age (greater than 34 years old), bad contact history, previously one of the retarded BH of monster, the past chromosome abnormalty youngster BH, man and wife chromosome abnormalities, B ultrasonic shows the pregnant woman of deformity, familial linkage inheritance medical history, consanguineous mating etc.Wherein, Women with Advanced Maternal Age is mainly to check object, has accounted at home 1/3 of whole inspection object.After cultivating, amniocyte by chromosome banding technique, can distinguish the disease of the karyomit(e) aspects such as trisome syndromes, balanced translocation, inversion and sex chromosomal abnormality.Because viable cell is few in the amniotic fluid, culture cycle is long, and aseptic requirement is high, does not slightly note namely causing failure.Even cultivate successfully, because of division mutually less, form is not good does not reach the analysis requirement, can't further study, and makes being applied to of amniotic fluid antenatal diagnosis restricted.Therefore successful amniocyte cultivation, except the strict experimental implementation technology and experience of needs, the composition of substratum also is very crucial.Amniocyte may be the cell that fetal skin, digestive tube, respiratory tract and urogenital tract come off, and wherein only having small portion is great-hearted cell (about 0.1%), and vigor cell is take adherent type cell as main.How urging its adherent propagation in vitro culture, is the key factor of successfully cultivating amniocyte.
Traditional amniotic fluid substratum forms: basic medium (RPMI1640, DMEM or F12)+calf serum (foetal calf serum).But the success ratio of this culture medium culturing is low, and success ratio is lower than 30%, and average culture cycle is long, and culture cycle needs 20 days, and division is few mutually, can not be effective to the amniocyte inspection; In order to shorten the amniocyte culture cycle, improve success ratio, had the research report of the substratum of modified form, be to add the extract that contains somatomedin on the basis of basic medium to form basically.Such as Chang HC (ChangHC, Jones OW, Masui H, Human amniotic fluid cells grown in ahormone-supplemented medium:suitability for prenatal diagnosis, Proc.Natl.Acad.Sci., Vol.79,4795-4799 (1982)).In the basic medium (DMEM/F12) that DMEM and F12 mixed by 1: 1, add 10 kinds of somatomedins, be respectively: Transferrins,iron complexes (transferrin) 5mg/L, Sodium Selenite 20nMol/L, Regular Insulin (insulin) 10mg/L, triiodothyronine (triiodothyronine) 0.1nMol/L, hyperglycemic-glycogenolytic factor (glucagon) 1mg/L, Prostatropin (bFGF) 10ug/L, hydrocortisone (hydrocortisone) 1nMol/L, testosterone (testosterone) 1nMol/L, estradiol (estradiol) 1nMol/L, progesterone (progesterone) 1nMol/L.Chang HC calls the H substratum to the substratum that contains these 10 kinds of somatomedin prescriptions, and the substratum after increasing these 10 kinds of somatomedin content doubly is called the H-1 substratum.The culture effect of the H-1 substratum that somatomedin doubles will be significantly better than the H substratum, H-1 is in the situation of the foetal calf serum of substratum interpolation lower aq, still can significantly increase cloning efficiency and the clonal growth speed of amniocyte, culture success ratio is reached more than 99%, and culture cycle shortened to about 10 days.
In the actual culturing process, although we can successfully turn out amniocyte by the H-1 substratum that forms in the basic medium basis interpolation commonly used such as DMEM/F12, α-MEM, F12 respectively that contains the different ratios foetal calf serum of discovery preparation, but it is lower still to exist clone's number, clonal expansion is slower, and the division phase cell of results is the problem such as less still.Therefore when the amniotic fluid samples of plantation was in poor shape, namely active cells was less, and clone's number only has 1-2, can't generate in a short time enough division phase cells, the cultivation of going down to posterity after need trysinization this moment, and this has prolonged cell culture period undoubtedly.Therefore the culture effect of H-1 substratum and the demand of Clinical Laboratory unit still have certain gap, are necessary culture medium prescription is improved.
Summary of the invention
The object of the present invention is to provide a kind of amniocyte clone's number and cell proliferation rate that can increase quickly and effectively in-vitro multiplication, shorten culture cycle, obtain many culture medium of amniotic fluid cells and the prescriptions thereof of division phase chromosome number.
The objective of the invention is to reach by following measures: studies show that, in cultivating, amniotic fluid increases antioxidant, reduce active oxygen ROS concentration, can promote clone and the propagation of amniocyte, in substratum, add the different types of antioxidant through optimum combination, reduce the active oxygen ROS concentration in the substratum, increase adherent type amniocyte clone number to reach, improve the synergistic action effect of cell proliferation rate.
A kind of culture medium of amniotic fluid cells is to comprise following component:
Basic medium DMEM/F12 cell culture medium
Transferrins,iron complexes (transferrin) 10mg/L
Sodium Selenite 40nMol/L
Regular Insulin (insulin) 20mg/L
Triiodothyronine (triiodothyronine) 0.2nMol/L
Hyperglycemic-glycogenolytic factor (glucagon) 2mg/L
Prostatropin (bFGF) 20ug/L
Hydrocortisone (hydrocortisone) 2nMol/L
Testosterone (testosterone) 2nMol/L
Estradiol (estradiol) 2nMol/L
Progesterone (progesterone) 2nMol/L
In order to improve the culture effect of culture medium of amniotic fluid cells, increase number of cell clones and divide mutually cell count, on the basis of above-mentioned component, add antioxidant.
Antioxidant is the mixture of vitamin-E and two kinds of antioxidants of L-AA.
The present invention adds the mixture of vitamin-E 5mg-20mg/L and L-AA 25-60mg/L.
The mixture that the present invention adds vitamin-E and L-AA has preferably effect, and vitamin-E and L-AA have significant synergy, can significantly increase the cell clone speed of growth and divide mutually cell count.And when vitamin-E be that 10mg/L, L-AA 50mg/L have best effect.
The present invention adds the foetal calf serum that volume percent is 4-15% in culture medium of amniotic fluid cells after, can be used for amniocyte cultivates, within 7, the 8 day short time, can cultivate a large amount of amniocyte that is in division stage that obtains to satisfy clinical diagnosis and scientific research requirement.
Amniocyte culture effect of the present invention has remarkable improvement, cloning efficiency and clonal growth speed obviously increase, reduced incubation time, improved simultaneously the cell cultures success ratio, given full play to the synergy of vitamin-E and the L-AA of different antioxidants, the synergy of mutually promoting of vitamin-E and L-AA, in the short period of time, can cultivate and obtain a large amount of amniocytes that is in division stage, can satisfy the requirement of clinical diagnosis and scientific research.
Embodiment
Embodiment 1: the culture medium culturing effect of different ingredients relatively
1, preparation H-1 substratum: the DMEM/F12 cell culture medium of 1L loading amount, after the injection water dissolving with 950ml, add NaHCO3 1.2g, 15mM HEPES, add 10 kinds of somatomedins, comprise Transferrins,iron complexes (transferrin) 10mg/L, Sodium Selenite 40nMol/L, Regular Insulin (insulin) 20mg/L, triiodothyronine (triiodothyronine) 0.2nMol/L, hyperglycemic-glycogenolytic factor (glucagon) 2mg/L, Prostatropin (bFGF) 20ug/L, hydrocortisone (hydrocortisone) 2nMol/L, testosterone (testosterone) 2nMol/L, estradiol (estradiol) 2nMol/L, progesterone (progesterone) 2nMol/L.Add injection water and be settled to 1000ml thereafter; Dispose 8 parts of H-1 substratum.
2, preparation DF-I1 substratum: get 1 part of H-1 substratum, add the vitamin-E of 20mg/L.
3, preparation DF-I2 substratum: get 1 part of H-1 substratum, add L-AA 60mg/L.
4, preparation DF-Ja substratum: get 1 part of H-1 substratum, add the vitamin-E of 5mg/L and the L-AA of 25mg/L.
5, preparation DF-Jb substratum: get 1 part of H-1 substratum, add the vitamin-E of 20mg/L and the L-AA of 60mg/L.
6, preparation DF-J1 substratum: get 1 part of H-1 substratum, add the vitamin-E of 10mg/L and the L-AA of 50mg/L.
7, preparation DF-I3 substratum: get 1 part of H-1 substratum, add the vitamin-E of 5mg/L.
8, preparation DF-I4 substratum: get 1 part of H-1 substratum, add L-AA 25mg/L.
9, the culture medium of amniotic fluid cells that respectively prepares being added volume percent is 4% foetal calf serum, becomes the amniocyte perfect medium.
7, amniocyte is cultivated: collect 10 parts of amniotic fluid samples (be the amniotic fluid samples of 17-22 between age in week, every part of about 2-2.5ml is altogether about 23ml), mix, obtain detecting with amniocyte and collect sample I, relatively the culture effect of different culture media.Cell cultures, flaking method carry out according to the following steps.
1. under aseptic condition, get each 2.5ml/ pipe of amniotic fluid, totally 8 pipes.
2. with centrifugal 10 minutes of 453g (1500rpm/min) room temperature.
3. suck supernatant liquor at aseptic operating platform, every pipe stays 1ml approximately, and suction pipe is broken up cell.Make cell suspension, add the 4ml substratum again and enter in the pipe, the light mixing of suction pipe moves to 25cm
2The mid-5%CO that contains of square batch cultur bottle
2The open cultivation of 37 ℃ of incubator saturated humidity row.
4. Microscopic observation after 6 days, as seen become fiber-like or epithelial cell growth, when Growth of Cells vigorous, when round cell appearred in the background of adherent layer, fresh amniotic fluid substratum 5ml was answered in commutation this moment, after 24-48 hour, good such as cell growth condition, add colchicine 0.04-0.08ug/ml, row cytology is processed about (20ug/ml, No. 7 syringe needles vertically erect and add 2) 4-6h.
5. harvested cell: pour out that enchylema enters the 10ml centrifuge tube in the bottle, twice of 0.85%NaCl solution flushing cell walls, 0.25% trysinization 3-5 minute, treat the cell face occur naked eyes as seen wrinkle shape when changing with centrifugal 10 minutes of lower cell 453g (1500rpm/min) room temperature of suction pipe piping and druming, remove supernatant, stay approximately 0.5ml.
6. hypotonic: add 37 ℃ of 0.075M KCl 4-6ml, suction pipe piping and druming, 37 ℃ water-bath 3-5 minute.
7. pre-fix: add 1.5ml methyl alcohol: Glacial acetic acid=3: 1 fixing agents, centrifugal 10 minutes of light 5 minutes 453g (1500rpm/min) of 37 ℃ of water-baths of mixing room temperature.Remove the honest and upright and thrifty 0.5ml of staying.
8. fixing: as to add 3: 1 fixing agent 8ml, light mixing, 37 ℃ of water-baths 10 minutes, centrifugal 10 minutes of 453g (1500rpm/min, eppendorf 5810R) room temperature.Remove supernatant, stay approximately 0.5ml.
9. repeat to fix: the same.Centrifugal 10 minutes of 453g (1500rpm/min) room temperature is removed supernatant, adds suitably several fixing agents depending on pipe floor cells amount.
10. drip sheet, roasting sheet: every 1-2 drips, and 75 ℃ of baking boxs toasted 3 hours.
Cultivate and finish to add up the cell clone sum before the chromosome sectioning, include clone's sum of division phase, and the size of effectively cloning; Statistics division phase cell number behind the preparation karyomit(e).
Table 1 substratum of respectively filling a prescription adds behind the tire ox blood to the culture effect of amniocyte relatively
Annotate:
1, the vitamin-E in each culture medium prescription of table 1 and L-AA concentration refer to add the front concentration of foetal calf serum.
2, the big or small judging criterion of clone is: in the observation that judges of 100 times of inverted microscopes, clone 〉=1 visual field and be judged as large clone; Clone≤1/2 visual field and then be judged as little clone; Marginal is medium clone.
3, always clone total clone's number of number=have division phase+nothing and divide clone's number of phase; Having division always to clone mutually number is the large, medium and small number sum of respectively cloning.
As can be seen from the results, on the H-1 medium base, add simultaneously the substratum of vitamin-E and L-AA, comprise DF-Ja, DF-Jb and DF-J1 and only add a kind of substratum (DF-I1 of antioxidant, DF-I2, DF-I3 and DF-I4) compare, total clone's number, cell number has obvious increase with dividing mutually to include the total clone's number that divides phase, illustrate that both have significant synergy to vitamin-E concentration when scope at 25-60mg/L of 5-20mg/L scope and L-AA concentration, and the vitamin-E in the DF-J1 substratum and L-AA concentration are optimal values, and best culture effect is arranged.The culture effect of the amniotic fluid substratum after optimizing has all had than the H-1 substratum of existing open source literature from total clone's number, division phase cell number, cell proliferation rate aspect and has increased substantially.
Embodiment 2: the culture medium of amniotic fluid cells preparation
1) material: Transferrins,iron complexes (transferrin), Regular Insulin (insulin), hyperglycemic-glycogenolytic factor (glucagon), triiodothyronine, hydrocortisone, hydrocortisone, male sex hormone, estradiol hormone, SIMGA company provides; The DM/F12 dehydrated medium, GIBCO company provides; Prostatropin (bFGF) lyophilized powder, Shanghai betting office provides; The reagent such as Sodium.alpha.-ketopropionate, Putriscine dihydrochloride, Sodium Selenite, VITAMIN, amino acid, deoxynucleoside and nucleosides, vitamin-E, L-AA, phenol red, glucose are all domestic analytical pure, and Guangzhou prestige reagent adding company provides;
2) equipment: osmotic pressure detector, pH value detector;
3) compound method
1. the preparation of 100X span amino acid mother liquor: the L-amino acid that substratum is required mixes (glutamine is preparation and freezing preservation separately, does not comprise wherein), prepares 100 times of span amino acid mother liquor 100ml.Amino acid whose kind and concrete weighing thereof are according to formula table 2, with load weighted amino acid dry powder blend, after adding 60ml water for injection, under magnetic agitation, slowly drip 1M HCl and dissolve fully to amino acid, inject water and be settled to 100ml and packing, 4 ℃ of preservations.
2. the preparation of (100X) VITAMIN mother liquor:, specifically fill a prescription according to formula table 2, with ten kinds support one's family be called usually join after, add in the water for injection in order.Every kind of VITAMIN needs to add another kind after the fully dissolving again, is made into 100X VITAMIN mother liquor 100ml, packing, 4 ℃ of preservations.Addition sequence is: D-VB5 calcium, i-inositol, niacinamide, pyridoxal hydrochloride, pyridoxine hydrochloride, thiamine hydrochloride, riboflavin, vitamin H (vitamin H), choline chloride 60, folic acid.
3. the preparation of L-glutaminate mother liquor: according to formula table 2, with after the L-glutaminate dry powder weighing of institute's expense with water for injection dissolving, packing ,-20 ℃ save backup.
4. the preparation of (100X) VB12 mother liquor: according to formula table 2, with after the independent weighing of VB12 of institute's expense with water for injection dissolving, packing, 4 ℃ of preservations.
5. the preparation of inorganic salt mother liquor: according to formula table 2, prepare respectively 100X calcium chloride mother liquor, copper sulfate mother liquor, iron nitrate mother liquor, ferrous sulfate mother liquor, Repone K mother liquor, magnesium chloride mother liquor, sal epsom mother liquor, sodium-chlor mother liquor, zinc sulfate and each 100ml of phosphoric acid salt mother liquor, normal temperature saves backup.
6. the preparation of Putriscine dihydrochloride mother liquor: according to formula table 2 preparation 100X Putriscine dihydrochloride mother liquor 100ml, 4 ℃ of preservations.
7. the preparation of 100X glucose mother liquid: according to formula table 2 preparation 100X glucose mother liquid 100ml, 4 ℃ of preservations after the packing.
8. the preparation of phenol red indicator mother liquor: according to the phenol red mother liquor 10ml of formula table 2 preparation 1000X, 4 ℃ of preservations after the packing.
9. 10ml (10
6X) preparation of hormone mother liquor: calculate hydrocortisone, hydrocortisone, male sex hormone, estradiol hormone dosage according to formula table 2, mix after the weighing respectively, with dehydrated alcohol (AR level) 10ml dissolving, be mixed with (10
6X) hormone mother liquor, 4 ℃ of preservations after the packing.
10. 10ml (10
6X) preparation of triiodothyronine mother liquor: calculate the triiodothyronine consumption according to formula table 2, with 1M NaOH 10ml dissolving, be mixed with 10
6X triiodothyronine mother liquor, 4 ℃ of preservations after the packing.
The preparation of 100ml (100X) L-AA mother liquor: calculate VITAMIN L-AA consumption according to formula table 2, with packing after the dissolving of 100ml water for injection, 4 ℃ keep in Dark Place.
4) culture medium of amniotic fluid cells adding volume percent is the foetal calf serum (FBS) of 4-15%, sterile filtration packing behind the mixing, the amniocyte perfect medium that obtains can be used for former generation or go down to posterity and cultivate ,-20 ℃ of preservations.
Table 2 culture medium of amniotic fluid cells prescription
* sodium chloride content can be according to the osmotic pressure value adjustment
Embodiment 3: the amniocyte perfect medium culture effect that contains different ratios foetal calf serum (FBS)
Collect 8 parts of amniocyte samples (be the amniotic fluid samples of 18-21 between age in week, every part of about 2-4ml is altogether about 22ml), mix, make to detect and collect sample with amniocyte.It is centrifugal after the amniocyte sample is divided into three parts, suck most of supernatant, stay the 1ml mixing to be inoculated in the culture medium of amniotic fluid cells that contains different FBS ratios of 4ml in embodiment 2 preparations the culture effect of relatively adding the amniocyte perfect medium of different volumes per-cent foetal calf serum.The results are shown in Table 3.
The amniocyte perfect medium culture effect that table 3 contains different ratios FBS compares
As can be seen from Table 3, not significantly difference of culture effect between the amniocyte perfect medium of the different percentage volume ratio foetal calf serums of interpolation all can be gathered in the crops a large amount of division phase amniocytes in a short time among the present invention.
Claims (4)
1. culture medium of amniotic fluid cells, by following component: basic medium, Transferrins,iron complexes, Sodium Selenite, Regular Insulin, triiodothyronine, hyperglycemic-glycogenolytic factor, Prostatropin, hydrocortisone, testosterone, estradiol, progesterone forms, and basic medium is the DMEM/F12 cell culture medium, wherein, basic medium is that DMEM mixes by 1 ﹕ 1 with F12, ten kinds of somatomedins content be respectively: Transferrins,iron complexes 10mg/L, Sodium Selenite 40nMol/L, Regular Insulin 20mg/L, triiodothyronine 0.2nMol/L, hyperglycemic-glycogenolytic factor 2mg/L, HBGH-2 0 μ g/L, hydrocortisone 2nMol/L, testosterone 2nMol/L, estradiol 2nMol/L, progesterone 2nMol/L is characterized in that on the basis of above-mentioned component, adds the mixture of vitamin-E and two kinds of antioxidants of L-AA.
2. a kind of culture medium of amniotic fluid cells according to claim 1 is characterized in that adding vitamin-E 5-20mg/L and L-AA 25-60mg/L.
3. a kind of culture medium of amniotic fluid cells according to claim 1 is characterized in that adding the mixture of vitamin e1 0mg/L and L-AA 50mg/L.
4. according to claim 1 and 2 or 3 described a kind of culture medium of amniotic fluid cells, it is characterized in that adding the foetal calf serum that volume percent is 4-15%.
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| CN102311938B (en) * | 2011-09-16 | 2013-11-06 | 南方医科大学珠江医院 | Serum-free medium for culturing hepatic cells |
| CN102618493A (en) * | 2012-04-13 | 2012-08-01 | 惠州鸿雨科技有限公司 | Culture medium for amniotic fluid and chorionic villus |
| CN105039244B (en) * | 2015-09-09 | 2017-07-21 | 广州和能生物科技有限公司 | A kind of low serum culture medium of amniotic fluid cells |
| CN106047798A (en) * | 2016-08-19 | 2016-10-26 | 上海逍鹏生物科技有限公司 | Aminotic cell culture medium for high-density cell culture system |
| CN106834215A (en) * | 2016-12-24 | 2017-06-13 | 严志海 | A kind of culture medium of amniotic fluid cells |
| CN108060118A (en) * | 2017-12-26 | 2018-05-22 | 深圳市龙岗区妇幼保健院 | The application of culture medium of amniotic fluid cells, amniotic fluid cell culture method and culture medium |
| CN109593707A (en) * | 2018-12-29 | 2019-04-09 | 广州和能生物科技有限公司 | A kind of culture medium of amniotic fluid cells and preparation method thereof |
| CN110747161B (en) * | 2019-11-28 | 2021-03-19 | 河南赛诺特生物技术有限公司 | Culture medium for amniotic fluid cells |
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